ML-265
(Synonyms: CID-44246499,NCGC00186528,TEPP-46) 目录号 : GC11741
A selective PKM2 activator
Cas No.:1221186-53-3
Sample solution is provided at 25 µL, 10mM.
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Quality Control & SDS
- View current batch:
- Purity: >98.00%
- COA (Certificate Of Analysis)
- SDS (Safety Data Sheet)
- Datasheet
| Cell experiment [1]: | |
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Cell lines |
661 W photoreceptor cell line |
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Preparation Method |
For 661 W cell line experiments, media was replaced prior to the start of treatment with ML-265. |
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Reaction Conditions |
Cells were incubated with10-7, 10-6, 10-5, 10-4, 10-3, 10-2 M concentrations of ML-265 for 2 hours |
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Applications |
ML-265 represents an ideal lead molecule to explore the neuroprotective effects of pharmacologically activating PKM2 in photoreceptors. This in vitro data suggests the small molecule activator is able to cross the cell membrane and enhance PK activity. |
| Animal experiment [1]: | |
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Animal models |
Brown-Norway mice |
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Preparation Method |
Adult rats were utilized for all in vivo ML-265 studies except ocular pharmacokinetic studies. All rats were housed at room temperature with 12-hour light and 12-hour dark cycle. |
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Dosage form |
Inject 2 μL of different concentrations（10-7, 10-6, 10-5, 10-4, 10-3, 10-2 M） of ML-265 slowly into the vitreous cavity，and harvest 4 hours later. |
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Applications |
Intravitreal injection of ML-265 was able to activate PK in vivo up to 170 ± 26% with an AC50 = 59 ± 38 μM. Considering both the specificity of ML-265 for PKM2 and the fact that PKM2 expression is confined to the outer retina, ML-265 is most likely able to traverse the retina and the cell membranes of photoreceptors to activate PKM2 。 |
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References: [1]. TJ Wubben, M Pawar, E Weh, et,al. Small molecule activation of metabolic enzyme pyruvate kinase muscle isozyme 2, PKM2,circumvents photoreceptor apoptosis. Sci. Rep., 10 (2020), p. 2990, doi.org /10.1038/s41598-020-59999-w. |
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ML-265 is widely used as an acknowledged PKM2 (pyruvate kinase muscle isoform 2) activator[1][2] ML-265 activates of PKM2 in both biochemical (AC50 = 92 nM) and cell-based assays with high selectivity over PKM1(pyruvate kinase muscle isoform 1), PKL and PKR. ML-265 was originally developed as a potential cancer therapy. Therefore, ML-265 represents an ideal lead molecule to explore the neuroprotective effects of pharmacologically activating PKM2[2]. PKM2, which is overexpressed in many cancers, is inhibited by ROS, allowing glycolytic flux to be shuttled into the oxidative PPP for NADPH generation. The small-molecule compounds, ML-265, can positively modulate PKM2, thereby decreasing glycolytic flux into the oxidative PPP and blunting NADPH biogenesis during ROS(fig.) [3].
ML-265 displayed powerful activation of recombinant human PKM2 with a maximum activation of 249 ± 14% (best-fit value ± std. error) , the half-maximum activating concentration (AC50) shows 108 ± 20 nM. To investigate the ability of ML-265 to activate PK in photoreceptors, the 661W cell line was utilized. Similar to the results observed with the recombinant enzyme, ML-265 increased PK activity in vitro (maximum activation = 515 ± 12% and AC50 = 19 ± 2 nM) [2].
To determine the intraocular pharmacokinetic profile of intravitreally administered ML-265 in rabbits. Single, intravitreal injections (50 μL) of two different doses of ML-265 were performed in rabbits. Assuming a vitreous volume of 1.15 mL, the final concentrations in the rabbit vitreous were approximately 100 μM and 1000 μM, respectively. The aqueous humor was sampled at multiple time points after the single intravitreal injection. The distribution phase has a half-life (t1/2) of 8.3 ± 0.5 hours. The elimination phase has a t1/2 = 141.6 ± 35 hours. ML-265 has a relatively long half-life in the eye following intravitreal injection and remains active at least two weeks after injection.[2].
References:
[1]: Walsh MJ, Brimacombe KR, Anastasiou D, et al. ML265: A potent PKM2 activator induces tetramerization and reduces tumor formation and size in a mouse xenograft model. Probe Reports from the NIH Molecular Libraries Program [Internet]
[2] TJ Wubben, M Pawar, E Weh, et,al. Small molecule activation of metabolic enzyme pyruvate kinase muscle isozyme 2, PKM2,circumvents photoreceptor apoptosis. Sci. Rep., 10 (2020), p. 2990, doi.org /10.1038/s41598-020-59999-w.
[3] Chakrabarti G, Gerber DE, Boothman DA. Expanding antitumor therapeutic windows by targeting cancer-specific nicotinamide adenine dinucleotide phosphate-biogenesis pathways. Clin. Pharmacol. Adv. Applications. 2015;7:57.
[4]: Xu W, Yang H, et,al. Oncometabolite 2-hydroxyglutarate is a competitive inhibitor of α-ketoglutarate-dependent dioxygenases. Cancer Cell. 2011 Jan 18;19(1):17-30. doi: 10.1016/j.ccr.2010.12.014. PMID: 21251613; PMCID: PMC3229304.
ML-265 被广泛用作公认的 PKM2（丙酮酸激酶肌肉亚型 2）激活剂[1][2] ML-265 激活 PKM2 在生物化学（AC50 = 92 nM）和基于细胞的测定中对 PKM1（丙酮酸激酶肌肉亚型 1）、PKL 和 PKR 具有高选择性。 ML-265 最初是作为一种潜在的癌症疗法而开发的。因此，ML-265 是探索药理激活 PKM2[2] 神经保护作用的理想先导分子。在许多癌症中过度表达的 PKM2 被 ROS 抑制，允许糖酵解通量穿梭到氧化 PPP 中以生成 NADPH。小分子化合物 ML-265 可以正向调节 PKM2，从而减少糖酵解通量进入氧化 PPP 并减弱 ROS 期间的 NADPH 生物合成（图）[3].
ML-265 显示重组人 PKM2 的强大激活，最大激活为 249 ± 14%（最佳拟合值 ± 标准误差），半数最大激活浓度（AC< sub>50) 显示 108 ± 20 nM。为了研究 ML-265 在光感受器中激活 PK 的能力，使用了 661W 细胞系。与用重组酶观察到的结果相似，ML-265 增加了体外 PK 活性（最大激活 = 515 ± 12% 和 AC50 = 19 ± 2 nM）[2].
确定玻璃体内注射 ML-265 在兔体内的眼内药代动力学特征。在兔子中进行了两种不同剂量的 ML-265 的单次玻璃体内注射 (50 μL)。假设玻璃体体积为 1.15 mL，兔玻璃体中的最终浓度分别约为 100 μM 和 1000 μM。在单次玻璃体内注射后，在多个时间点对房水进行取样。分布相的半衰期 (t1/2) 为 8.3 ± 0.5 小时。消除阶段的 t1/2 = 141.6 ± 35 小时。 ML-265 在玻璃体内注射后在眼中具有相对较长的半衰期，并且在注射后至少两周仍保持活性。[2]。
| Cas No. | 1221186-53-3 | SDF | |
| 别名 | CID-44246499,NCGC00186528,TEPP-46 | ||
| 化学名 | 6-[(3-aminophenyl)methyl]-4,6-dihydro-4-methyl-2-(methylsulfinyl)-5H-thieno[2’,3’:4,5]pyrrolo[2,3-d]pyridazin-5-one | ||
| Canonical SMILES | CS(C1=CC(N2C)=C(S1)C3=C2C(N(CC4=CC(N)=CC=C4)N=C3)=O)=O | ||
| 分子式 | C17H16N4O2S2 | 分子量 | 372.5 |
| 溶解度 | ≥ 37.3mg/mL in DMSO | 储存条件 | Store at -20°C |
| General tips | 请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；一旦配成溶液，请分装保存，避免反复冻融造成的产品失效。 储备液的保存方式和期限：-80°C 储存时，请在 6 个月内使用，-20°C 储存时，请在 1 个月内使用。 为了提高溶解度，请将管子加热至37℃，然后在超声波浴中震荡一段时间。 |
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| Shipping Condition | 评估样品解决方案：配备蓝冰进行发货。所有其他可用尺寸：配备RT，或根据请求配备蓝冰。 | ||
| 制备储备液 | |||
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1 mg | 5 mg | 10 mg |
| 1 mM | 2.6846 mL | 13.4228 mL | 26.8456 mL |
| 5 mM | 0.5369 mL | 2.6846 mL | 5.3691 mL |
| 10 mM | 0.2685 mL | 1.3423 mL | 2.6846 mL |
| 第一步：请输入基本实验信息（考虑到实验过程中的损耗，建议多配一只动物的药量） | ||||||||||
| 给药剂量 | mg/kg |  |
动物平均体重 | g | |
每只动物给药体积 | ul | |
动物数量 | 只 |
| 第二步：请输入动物体内配方组成（配方适用于不溶于水的药物；不同批次药物配方比例不同，请联系GLPBIO为您提供正确的澄清溶液配方） | ||||||||||
| % DMSO % % Tween 80 % saline | ||||||||||
| 计算重置 | ||||||||||
计算结果:
工作液浓度： mg/ml；
DMSO母液配制方法： mg 药物溶于 μL DMSO溶液（母液浓度 mg/mL，
体内配方配制方法：取 μL DMSO母液，加入 μL PEG300，混匀澄清后加入μL Tween 80，混匀澄清后加入 μL saline，混匀澄清。
1. 首先保证母液是澄清的；
2.
一定要按照顺序依次将溶剂加入，进行下一步操作之前必须保证上一步操作得到的是澄清的溶液，可采用涡旋、超声或水浴加热等物理方法助溶。
3. 以上所有助溶剂都可在 GlpBio 网站选购。
Pharmacological Activation of Pyruvate Kinase M2 Inhibits CD4 + T Cell Pathogenicity and Suppresses Autoimmunity
Cell Metab2020 Feb 4;31(2):391-405.e8.PMID: 31761564DOI: 10.1016/j.cmet.2019.10.015
Pyruvate kinase (PK) catalyzes the conversion of phosphoenolpyruvate to pyruvate during glycolysis. The PK isoform PKM2 has additional roles in regulation of gene transcription and protein phosphorylation. PKM2 has been shown to control macrophage metabolic remodeling in inflammation, but its role in T cell biology is poorly understood. Here, we report PKM2 upregulation, phosphorylation, and nuclear accumulation in murine and human CD4+ T cells following activation in vitro. Treatment of T cells with TEPP-46, an allosteric activator that induces PKM2 tetramerization and blocks its nuclear translocation, strongly reduces their activation, proliferation, and cytokine production by inhibiting essential signaling pathways and thus preventing the engagement of glycolysis. TEPP-46 limits the development of both T helper 17 (Th17) and Th1 cells in vitro and ameliorates experimental autoimmune encephalomyelitis (EAE) in vivo. Overall, our results suggest that pharmacological targeting of PKM2 may represent a valuable therapeutic approach in T cell-mediated inflammation and autoimmunity.
Targeting Pyruvate Kinase M2 Phosphorylation Reverses Aggressive Cancer Phenotypes
Cancer Res2021 Aug 15;81(16):4346-4359.PMID: 34185676DOI: 10.1158/0008-5472.CAN-20-4190
Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype with low survival rate and a lack of biomarkers and targeted treatments. Here, we target pyruvate kinase M2 (PKM2), a key metabolic component of oncogenesis. In patients with TNBC, PKM2pS37 was identified as a prominent phosphoprotein corresponding to the aggressive breast cancer phenotype that showed a characteristic nuclear staining pattern and prognostic value. Phosphorylation of PKM2 at S37 was connected with a cyclin-dependent kinase (CDK) pathway in TNBC cells. In parallel, pyruvate kinase activator TEPP-46 bound PKM2pS37 and reduced its nuclear localization. In a TNBC mouse xenograft model, treatment with either TEPP-46 or the potent CDK inhibitor dinaciclib reduced tumor growth and diminished PKM2pS37. Combinations of dinaciclib with TEPP-46 reduced cell invasion, impaired redox balance, and triggered cancer cell death. Collectively, these data support an approach to identify PKM2pS37-positive TNBC and target the PKM2 regulatory axis as a potential treatment. SIGNIFICANCE: PKM2 phosphorylation marks aggressive breast cancer cell phenotypes and targeting PKM2pS37 could be an effective therapeutic approach for treating triple-negative breast cancer.
The PKM2 activator TEPP-46 suppresses kidney fibrosis via inhibition of the EMT program and aberrant glycolysis associated with suppression of HIF-1α accumulation
J Diabetes Investig2021 May;12(5):697-709.PMID: 33314682DOI: 10.1111/jdi.13478
Aims/introduction: Tubulointerstitial fibrosis is a hallmark of diabetic nephropathy and is associated with an epithelial-to-mesenchymal transition (EMT) program and aberrant glycolysis. Dimeric pyruvate kinase (PK) M2 (PKM2) acts as a key protein kinase in aberrant glycolysis by promoting the accumulation of hypoxia-inducible factor (HIF)-1α, while tetrameric PKM2 functions as a pyruvate kinase in oxidative phosphorylation. The aim of the research is to study the effect of PKM2 tetramer activation on preventing kidney fibrosis via suppression of aberrant glycolysis and the EMT program.
Materials and methods: In vivo: Streptozotocin (STZ) was utilized to induce diabetes in 8-week-old CD-1 mice; 4 weeks after diabetes induction, proteinuria-induced kidney fibrosis was developed by intraperitoneal injection of bovine serum albumin (BSA: 0.3 g/30 g BW) for 14 days; The PKM2 activator TEPP-46 was also administered orally simultaneously. In vitro: HK2 cells were co-treated with high-glucose media or/and TGF-β1 and TEPP46 for 48 h, cellular protein was extracted for evaluation.
Results: Diabetic mice developed kidney fibrosis associated with aberrant glycolysis and EMT; BSA injection accelerated kidney fibrosis in both the control and diabetic mice; TEPP-46 rescued the kidney fibrosis. In HK2 cells, TEPP-46 suppressed the EMT program induced by TGF-β1 and/or high-glucose incubation. TEPP-46-induced PKM2 tetramer formation and PK activity resulted in suppression of HIF-1α and lactate accumulation. Specific siRNA-mediated knockdown of HIF-1α expression diminished high glucose-induced mesenchymal protein levels.
Conclusion: PKM2 activation could restore the tubular phenotype via suppression of the EMT program and aberrant glycolysis, providing an alternative target to mitigate fibrosis in diabetic kidneys.
Tetrameric PKM2 Activation Curbs CD4 + T Cell Overactivation
Trends Endocrinol Metab2020 Jun;31(6):393-395.PMID: 32340764DOI: 10.1016/j.tem.2020.04.001
Angiari et al. recently reported that TEPP-46 induces PKM2 tetramerization, thereby inhibiting its nuclear translocation and suppressing CD4+ T cell activation, T helper (Th)1/Th17 cell development, and experimental autoimmune encephalomyelitis (EAE) development both in vitro and in vivo. Moreover, TEPP-46 suppresses T cell glycolysis. These findings identify PKM2 tetramerization as a potential therapeutic target.
Small molecule activation of metabolic enzyme pyruvate kinase muscle isozyme 2, PKM2, circumvents photoreceptor apoptosis
Sci Rep2020 Feb 19;10(1):2990.PMID: 32076076DOI: 10.1038/s41598-020-59999-w
Photoreceptor cell death is the ultimate cause of vision loss in many retinal disorders, and there is an unmet need for neuroprotective modalities to improve photoreceptor survival. Similar to cancer cells, photoreceptors maintain pyruvate kinase muscle isoform 2 (PKM2) expression, which is a critical regulator in aerobic glycolysis. Unlike PKM1, which has constitutively high catalytic activity, PKM2 is under complex regulation. Recently, we demonstrated that genetically reprogramming photoreceptor metabolism via PKM2-to-PKM1 substitution is a promising neuroprotective strategy. Here, we explored the neuroprotective effects of pharmacologically activating PKM2 via ML-265, a small molecule activator of PKM2, during acute outer retinal stress. We found that ML-265 increased PKM2 activity in 661 W cells and in vivo in rat eyes without affecting the expression of genes involved in glucose metabolism. ML-265 treatment did, however, alter metabolic intermediates of glucose metabolism and those necessary for biosynthesis in cultured cells. Long-term exposure to ML-265 did not result in decreased photoreceptor function or survival under baseline conditions. Notably, though, ML-265-treatment did reduce entrance into the apoptotic cascade in in vitro and in vivo models of outer retinal stress. These data suggest that reprogramming metabolism via activation of PKM2 is a novel, and promising, therapeutic strategy for photoreceptor neuroprotection.